Project Summary Shigella is a genus of pathogenic bacteria responsible for over 165 million infections and 1.1 million deaths each year. Our recent studies have demonstrated that RNA thermometers (RNATs) control the expression of several virulence-associated genes in this important pathogen. The current definition of an RNAT is a simple one: a temperature-sensitive structural element located within the 5' UTR of the regulated transcript that blocks ribosomal binding at non-permissive temperatures. Defined as the minimal sequence unit sufficient to confer such regulation, it is generally assumed that the mechanism of RNAT-mediated regulation is conserved and that it is independent of additional cellular factors. Work by us and others have drawn this simple definition into question. It has become abundantly clear that RNATs are a diverse collection of ribo-regulators, differing in size, structure, location within a regulated transcript and the cellular processes that they control. Our central hypothesis is that the structure and regulatory activity of different RNATs respond differently to uniform changes in temperature, and that such responses are influenced by additional sequences within the 5' UTR and/or binding of rRNA. Such potential complexities of RNAT activity have not been investigated. To this end, the overall goal of this study is to complete comprehensive genetic and biochemical characterizations of four unique virulence- associated RNATs located within virulence-associated genes of S. dysenteriae. The overall goal of this application will be achieved in three specific aims. Aim 1 will measure the differential effect of temperature on the structure of different RNATs and identify the impact of additional 5' UTR sequences and rRNA on such responses. Aim 2 will determine the differential effect of temperature on ribosomal binding to different RNATs and measure the impact of additional 5' UTR sequences on this response. Aim 3 will characterize the impact of each RNAT on specific S. dysenteriae virulence-associated processes. The multi-disciplinary analysis of four unique RNATs from a single bacterial pathogen is a powerful and novel approach with the strong potential to contribute foundational knowledge to the field. In addition, the proposed studies will make a significant contribution to the current understanding of Shigella virulence. The complementary skill sets of the PI (Murphy; RNATs, bacterial pathogenesis) and Co-Investigator (Hines; RNA structure-function, biochemistry) ideally position this team to successfully complete this multidisciplinary study, a study that will allow the research training of twelve undergraduate students, two graduate students and three medical student researchers.